This invention relates to hybrid compounds which contain a portion of Interleukin 2 (IL -2) including a region of the IL -2 receptor (IL-2R) binding domain.
Certain hybrid compounds falling within the foregoing classification are known to be of diagnostic and therapeutic value. In particular, a class of hybrid proteins of significant therapeutic value (e.g., for prevention of allograft rejection) contains the following protein segments joined together by peptide bonds: (a) the enzymatically active Fragment A of diphtheria toxin, (b) a segment including the cleavage domain adjacent Fragment A, (c) a segment including at least a portion of the hydrophobic domain of Fragment B of diphtheria toxin and not including the generalized eukaryotic binding domain of Fragment B, and (d) a segment including a portion of IL-2 which includes a region of the IL-2R binding domain of IL-2 effective to cause the hybrid protein to bind selectively to and kill a predetermined class of cells which bear an IL-2R. This class of proteins is referred to herein as DAB IL-2. (Murphy U.S. Pat. No. 4,675,382, hereby incorporated by reference.) It is important to be able to obtain highly purified preparations of IL-2-containing hybrid molecules such as DAB-IL-2 molecules in order to avoid any possible harmful effects associated with impurities in such preparations.
It is known that IL-2 can be easily purified on hydrophobic reverse phase columns. However, DAB IL-2 binds irreversibly to such resins, which therefore cannot be used to purify these hybrid proteins. There are other major differences which have been demonstrated between the binding properties of IL-2 and DAB IL-2. For instance, DAB-IL-2 typically binds with 5 10 fold lower affinity to the high affinity IL-2 receptor, as compared to IL-2 In addition, DAB-IL-2 molecules form complex intermolecular covalent (disulfide) and non covalent bonds among themselves and with the major DAB IL-2 subfragments (59 kD, 49 kD, and 47 kD) produced in E. coli lysates, resulting in heterogeneous oligomeric structures, e.g., 68-68 dimers, 68-49 dimers, 68-47 dimers, and 68-49-47 trimers, among others.
A recent publication by Sherblom et al., J. Immunol. 143:939-944 (1989) demonstrated that recombinant IL-2 acts as a lectin that binds specifically to a complex high mannose carbohydrate sequence found on several glycoproteins, including hen egg-white ovalbumin and human uromodulin. This high mannose sequence (referred to as M5[6], and illustrated diagrammatically in FIG. 4) has a branched bi arternary mannose component attached to a core of diacetylchitobiose. Sherblom et al., using amino acid homoloqy comparison to known lectin binding regions, hypothesized that the carbohydrate binding site of IL-2 is near its amino terminal end; it is the amino terminal end of IL-2 that is fused to the diphtheria toxin derived sequences in DAB-IL-2.